Taxol production by a microbe

ABSTRACT

Taxol is produced from taxol-producing micro-organisms. Methods of  obtain the taxol-producing microorganisms are described. Radioactive labelled taxol products and methods for use of the radioactive labelled taxol and for the treatment of leukemia and cancer cells are described.

This invention was made with support under Grant No. DHE-9206803 (NSF)awarded by National Science Foundation.

This application is a divisional of application Ser. No. 08/258,105,filed Jun. 10, 1994, now U.S. Pat. No. 5,861,302 which is a continuationin part application of Ser. No. 07/971,508 filed Nov. 4, 1992, now U.S.Pat. No. 5,322,799, issued Jun. 21, 1994, which is a continuation inpart of application Ser. No. 07/869,726, filed Apr. 16, 1992, nowabandoned.

TECHNICAL FIELD

The present invention relates to the use of one or more microorganismsto produce taxol (and related taxanes). The invention discloses themethod of the discovery of said microorganisms, their isolation,screening for taxol production, growth requirements for taxolproduction, and chemical evidence for taxol (taxane) production.

BACKGROUND OF THE INVENTION

Taxol, which is of the chemical structural formula: ##STR1## showssignificant properties of promoting the polymerization of tubulin andinhibiting the depolymerization of microtubules. For these reasons,taxol is a valuable antileukemic and antitumor agent and is the subjectof increasing research.

Taxol is known to be extracted from the trunk bark of different speciesof the Taxus, or Yew tree. Yields are generally low, usually on theorder of no more than about 100 milligrams per kilogram in theextraction process. Various procedures for the production of taxol areknown for example, from U.S. Pat. Nos. 4,814,470 and 4,857,653. Achemical process for the preparation of taxol is disclosed in U.S. Pat.No. 4,924,011.

Wani et al, "Journal of the American Chemical Society", Vol. 93, May1971, No. 9, pages 2325-2327, reports on the structure of taxol and itspotential use as an antileukemic and tumor inhibitory compound. Thispublication further discusses an alcohol extraction procedure forobtaining taxol from the stem bark of the western Yew tree (Taxusbrevifolia).

The Pacific yew, Taxus brevifolia, is one of only ten Taxus speciesknown worldwide. It is not confined to the Pacific coast of NorthAmerica as its name might imply, but grows inland as far east as GlacierNational Park, Montana. Generally, it is a small tree, 7-13 meters inheight and 5-10 cm in diameter. The crown is large and conical.Commonly, however, it is contorted with the main stem and some of thelower limbs growing close to the ground producing numerous adventitiousroots resulting in a complex and dense interwoven thicket of growth. Thetree is usually associated with deep, rich, moist soils near streams andlakes. It is an understory tree commonly found with Douglas fir, Westernhemlock, Western red cedar and Western larch.

The inner bark of this remarkable little tree is the primary source oftaxol. Taxol is a highly derivatized terpenoid having the structureindicated above, and has shown remarkable promise as an anti-tumor agentespecially in breast and ovarian cancers. Unfortunately, at the presenttime, the supplies of taxol are inadequate to meet the current orprojected demands. Taxol is only currently available from extract fromthe bark of yew trees. The inadequate supply of taxol is reflected inits current market price which is $6000.00 per gram. Thus, it isessential to understand how, where, and when, taxol is biosynthesized inthe tree and the factors that affect its biosynthesis.

It is likely that many factors influence the production of taxol byPacific yew. These include not only various environmental factors suchas temperature and moisture level, but the genetic background of thetree itself. Also, plants are commonly hosts to a multitude of microbesincluding parasites, symbionts, endophytes, epiphytes, and mycorrhizalfungi. These organisms may also influence the production of secondaryplant metabolites such as phytoalexins, whose presence can be triggeredby elicitors from microbes. Such microbes may catabolize or derivatizeplant compounds.

These and other reasons prompted the present inventors to devise an "invitro" system of taxol production (see related U.S. patent applicationSer. No. 07/845,097, filed Mar. 3, 1992. The system utilizes isotopicprecursors of taxol, an optimized environment and the appropriate plantparts where taxol is synthesized. The result was an "in vitro" system oftaxol synthesis from the most productive tissue portions of the Pacificyew tree.

However, the above in vitro synthesis described in related applicationSer. No. 07/845,097 has certain limitations. The source of taxolproduction, the Pacific yew, is a relatively rare tree, and there isconcern that the supply of taxol is not adequate to meet the demand.

Moreover, other methods, including total chemical synthesis, andderivatization of baccatin to yield taxotere are both inadequate. Thechemical synthesis methods are multi-stepped and non-economicallyfeasible while the taxane derivatization method utilizes a taxaneisolated from yew needles.

Clearly, a microbial source of taxol would be preferable if it could beeasily grown, would produce taxol (or a related taxane), and utilize theenormous U.S. biotechnology industry fermentation capabilities.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide animproved process for producing taxol, using a taxol producing microbefollowed by separation of taxol from the growth medium and/or microbe.

In a further embodiment of the invention, there is provided aradioactive labelled taxol product and methods for use of theradioactive labelled taxol. The labelled taxol can be produced by use ofa labelled precursor as described herein. Radiolabelled taxol is a newcompound for the present invention and related application Ser No.07/845,097 (which produces the labelled taxol by a different process).Because of its radiolabel, it and its derivatives can be identified inthe mammal body so one can determine how it functions as an antileukemicand antitumor agent. The taxol may be labelled with any label (stable orunstable) including ¹⁴ C, ¹³ C, tritium (³ H) or with ¹⁵ N.

STATEMENT OF THE INVENTION

The present invention provides an improved process for producing taxol,which uses a taxol producing microorganism followed by separation oftaxol from the growth medium and/or microorganism. One aspect of theinvention is a method for isolating a microorganism which produces ataxane, which comprises the following steps:

(a) obtaining tissue fragments from a tree of the Genus Taxus,

(b) placing said tissue fragments on agar medium until fungal growthoccurs e.g., about 2-5 days,

(c) placing fungal hyphae from said fungal growth on mycological agar,and replacing said fungal hyphae on said mycological agar if necessary,until a culture in pure form is obtained,

(d) transferring said fungal hyphae to a fungal lab growth medium, withsubsequent growth of the fungal culture,

(e) removing at least a portion of the culture media containing thefungal culture, thoroughly grinding the mycelium, and adding an organicsolvent to the mixture,

(f) obtaining a chromatograph of said fungal culture in said solvent,

(g) checking the solution for the taxane reaction, e.g., withvanillin-sulfuric acid spray, and comparing the chromatograph with oneor more taxane standards such as taxol, baccatin, cephalomannine, andoptionally

(h) discarding the cultures which do not produce taxol. Optionally, thecultures which do not produce taxol may be preserved for additionaltesting.

Preferred members of the Genus Taxus are Taxus brevifolia, Taxusbaccata, Taxus cuspiduta, Taxus canadensis, and Taxus floridana.Particularly preferred is Taxus brevifolia.

The present invention further provides a class of microbes which havetaxol-producing characteristics. Montana BA, the characteristics ofwhich are described in detail below, is representative of such microbes.Ten additional taxol producing microbes are also disclosed. The microbesaccording to the present invention produce taxol in culture. Preferredtaxol-producing microbes are fungi, and particularly preferred is ataxol producing fungi isolated from a yew tree. Even more preferred is afungus designated Montana BA. The present invention provides the majordiscovery of microbes which will produce taxol. The invention covers anymicrobe which has taxol producing characteristics. The specific microbesdescribed are considered representative only.

Also, the present invention comprises a taxane composition obtained byculturing a microbe. Preferred is a taxane of the species taxol.Particularly preferred is a taxol composition produced by a microbe,e.g., a fungus having the taxol-producing characteristics of Montana BA.

In another aspect, the present invention provides a radiolabelled taxanecomposition obtained by culturing a microbe. Preferred is aradiolabelled taxol composition produced by a microbe which is a fungus.Even more preferred is a labelled composition produced by a microbewhich is a fungus having the characteristics of Montana BA. Particularlypreferred labels are ¹⁴ C, ¹³ C, ³ H, or ¹⁵ N.

The present invention provides an improved method for producing a bulkpharmaceutical composition, which contains a pharmaceutically effectiveamount of a taxol composition, combined with one or morepharmaceutically acceptable inert or physiologically active diluents oradjuvants.

Also provided is a pharmaceutical composition, which contains apharmaceutically effective amount of a radiolabelled taxol composition,as described above, combined with one or more pharmaceuticallyacceptable inert or physiologically active diluents or adjuvants.

Moreover, the present invention includes a method for the treatment ofleukemia or tumors which comprises administering a pharmaceuticalcomposition containing taxol as described above.

In yet another aspect, the present invention provides a method forproducing a taxane, which comprises

a) exposing a taxane producing microbe according to the presentinvention as described above to a nutrient media capable of supportinggrowth of the microbe,

b) providing culturing conditions for the media containing the microbe,which conditions are capable of producing growth and reproduction of themicrobe, and

c) isolating or concentrating the desired taxane from said culture mediaor said microbe.

Preferred is a method for producing a taxol composition wherein themicrobe has the taxol-producing characteristics of Montana BA. Morepreferred is such a method wherein the microbe is a taxol producingfungi. Further preferred is a method wherein the microbe is isolatedfrom a yew tree.

Also preferred is a method for producing a taxane, as described above,wherein the nutrient media comprises benzoic acid, a benzoic acidmetabolite precursor, or a salt of benzoic acid, such as sodiumbenzoate. Particularly preferred is a method as described above forproducing the taxane, wherein the taxane is taxol.

A preferred method for producing a taxane comprises

a) exposing a taxol producing microbe to a nutrient media capable ofsupporting growth of said microbe,

b) providing culturing conditions for said media containing saidmicrobe, which are capable of producing growth and reproduction of saidmicrobe, and

c) isolating or concentrating said taxane from said culture media orsaid microbe.

Also preferred is a method for producing a taxane which comprises

a) exposing a taxol producing microbe of the invention to a nutrientmedia capable of supporting growth of said microbe, wherein said mediacontains benzoic acid,

b) providing culturing conditions for said media containing saidmicrobe, which conditions are capable of producing growth andreproduction of said microbe, and

c) isolating or concentrating said taxane from said culture media orsaid microbe.

In another preferred aspect of the present invention provides a methodfor producing a taxol which comprises

a) exposing a taxol producing microbe of the invention to a nutrientmedia capable of supporting growth of said microbe, wherein said mediacontains benzoic acid,

b) providing culturing conditions for said media containing saidmicrobe, which conditions are capable of producing growth andreproduction of said microbe, and

c) isolating or concentrating said taxol from said culture media or saidmicrobe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the NMR spectrum of a semi-purified culture extract fromMontana BA fungus.

FIG. 2 shows a FAB mass spectrum of taxol obtained by culturing theMontana BA fungus.

FIG. 3 shows a flow injection ion spray mass spectrum of TLC fraction RF0.18 of fungus extract after injections of diluent wherein the lowintensity ions consistent with taxol are m/z 854 and 871.

FIG. 4 shows a scanning electron micrograph of hyphae and fructigenoushyphae of T. andreanae; Bar equals 10 μm.

FIG. 5 shows a scanning electron micrograph of a series of various shapebulbils of T. andreanae; Bar=10 μm.

FIG. 6 shows a scanning electron micrograph of single bulbil of T.andreanae illustrating the organization of the cells of the bulbil;Bar=10 μm.

FIG. 7 shows the growth of T. andreanae on the inner bark of Pacific yew(left) and growth and bulbil formation on the leaves in small lymphfragments of T. andreanae (right).

FIG. 8 shows the transmission electron micrograph of bulbil cells of T.andreanae illustrating a dense cytoplasm in each cell; Bar=1 μm.

FIG. 9 shows the transmission electron micrograph of T. andreanae bulbilcells at a higher magnification. Note that the cell wall has twodistinct layers, and a fibrous material between the cells; Bar=1 μm.

FIG. 10 shows strain 1ND 100× magnified by electron microscope.

FIG. 11 shows strain H10BA2 100× magnified by electron microscope.

FIG. 12 shows strain WIC65NC 100× magnified by electron microscope.

FIG. 13 shows strain CC45BD 100× magnified by electron microscope.

FIG. 14 shows strain CC50NA1 100× magnified by electron microscope.

FIG. 15 shows strain CC54BE 100× magnified by electron microscope.

FIG. 16 shows strain CC64BB 100× magnified by electron microscope.

FIG. 17 shows strain CC54BA 100× magnified by electron microscope.

FIG. 18 shows strain CC53NA-2 100× magnified by electron microscope.

FIG. 19 shows strain CC52NC 100× magnified by electron microscope.

DESCRIPTION OF THE INVENTION

All land plants serve as life support systems to a host of microbes. Themicrobes exist as parasites, saprophytes, endophytes, symbionts ormycorrhizae. On occasion, there may be intergeneric exchange of geneticinformation between one or more of these organisms and its hosts, orvise versa. With the former case, it is well documented that certainspecies of agrobacteria are capable of genetically transforming theirhost plants via either the Ri or Ti plasmids. The result is that agenetically altered plant is formed that has one or more characteristicsof the agrobacterium.

Numerous cases are known in which all phytohormones, known throughoutthe plant kingdom, are also produced by one or more plant parasiticbacteria or fungi. These include indole acetic acid, the gibberillins,some cytokinins, abscissic acid and ethylene. Recently, the presentapplicants have also shown that certain terpenoids that were originallythought to be exclusively higher plant associated compounds, are alsoproduced by a plant pathogenic fungus (See, Bunkers et al., "Productionof Petasol", Mycol. Res 95, (3); 347-351 (1991)).

Thus, while there is no published evidence that taxol could or would beproduced by any microorganism, the present applicants explored themicroorganism possibility. The inventors suspected that taxol (taxanes)may be produced by one or more microbes associated with the yew tree.

The basis for this concept was that microbes may exist which willproduce taxol because of a possible genetic exchange which may havepreviously occurred, either between the microbe(s) (as original sourceof taxol) or yew (as original source of taxol). The net result would bethe most desirable case of possessing one or more microbes which couldbe placed in fermenters to produce taxol.

The present invention provides a method for locating and isolatingmicroorganisms which produce taxol, and provides specific examples wheresuch taxol producing organisms are found. Other microorganismsassociated with the yew or related trees, which produce taxol or relatedtaxanes, may be isolated using the present method. Therefore, thepresent invention is considered to be inclusive of all such microbes, orany pharmaceutical.

One method of the invention involves finding, isolating andcharacterizing one or more microbes from yew forests or elsewhere whichproduce taxol (taxanes). One place to focus the search for such anorganism was in one or more locations which have naturally supported thegrowth of yew trees for centuries.

After locating the yew tree, the following general method is followed toisolate microbes which produce taxol. Small stems were cut from the yewtree, treated with 70% ethanol as a disinfectant and then, with asterile blade, removing the outer bark. Pieces of the inner bark of thestem, which is enriched with taxol (Wani's paper), are then placed onagar medium (water agar) until fungal growth occurs after 2-5 days.Then, tips of the fungal hyphae are removed from the water agar andsubsequently placed on mycological agar. The culture is obtained in apure form as judged by its behavior on the plate. Lab contaminantsseldom occur due to the rigor of the aseptic technique which was used.

Once the organism is obtained in a pure form it is ultimatelytransferred to one or more lab media. In this case the modified M-1-Dmedium (Table 1, below) with yeast extract is used to support the growthof the microbe. Alternatively, (Table 2, below) another medium whichresembles the soluble sugars (quantity and quality) of the yew bark,plus a mixture of amino acids (or critical amino acids and anothernitrogen source, such as ammonium salts), Na benzoate and acetate areincluded.

                  TABLE 1                                                         ______________________________________                                        Modified M-I-D Media (Filner)                                                 ______________________________________                                                                    g/l                                               ______________________________________                                          Ca(NO.sub.3).sub.2 1.20 mM 0.28                                               KNO.sub.3 0.79 mM 0.08                                                        KCl 0.87 mM 0.06                                                              MgSO.sub.4 3.00 mM 0.36                                                       NaH.sub.2 PO.sub.4 --H.sub.2 O 0.14 mM 0.02                                   Sucrose 87.60 mM  30.00                                                       Ammonium Tartrate 27.10 mM  5.00                                            ______________________________________                                            mg/l                                                                      ______________________________________                                          FeCl.sub.3 --6 H.sub.2 O 7.4 μM 2.0                                        MnSO.sub.4 30.0 μM  5.0                                                    ZnSO.sub.4 --7 H.sub.2 O 8.7 μM 2.5                                        H.sub.3 BO.sub.3 2.2 μM 1.4                                                KI 4.5 μM 0.7                                                              pH 5.5 with 0.1 M HCl                                                         0.25 g Yeast Extract*                                                       ______________________________________                                         *Or omit the Yeast and supplement with:                                       Stock Biotin 0.5 mg/ml                                                        " Thiamine 0.5 mg/ml in 40% aq. EtoH                                          " Inositol 5 mg/ml                                                            Use 2 ml/l of broth                                                      

                  TABLE 2                                                         ______________________________________                                        Taxol Microbial Culture Medium                                                                   Grams/Liter                                                ______________________________________                                        glucose            1                                                            fructose 3                                                                    sucrose 6                                                                     KHPO.sub.4 --KH.sub.2 PO.sub.4 1 ml of 1M pH 6.8                              MgSO.sub.4 .36                                                                Ca(NO.sub.3).sub.2 H.sub.2 O .65                                              Yeast extract 0.5 g                                                         ______________________________________                                          Ca(NO.sub.3).sub.2 1.0 mg                                                     ZnSO.sub.4 2.5                                                                MnCl.sub.2 .5                                                                 FeCl.sub.2 2.0                                                                leucine 0.1 mM                                                                phenylalanine 0.01 mM                                                         NaAc 1.0 mM                                                                 ______________________________________                                    

The incubation of the fungus is carried out at 25° C. (in 100 ml ofmedium in a 250 ml flask, for example) under still conditions(occasionally shake periodically--every other day, for example) for 3weeks. At the end of the incubation period the fluid (media) is decantedand the mycelium is thoroughly ground (disrupted) at maximum speed in aSorvall Ominimixer for 30 sec. Then, an equal volume of chloroformmethanol solution 10:1 v/v is added to the medium and ground fungalmycelial solutions. The chloroform layer (bottom) is removed in aseparatory funnel. The process may be twice repeated. Ultimately, thechloroform layers are collected together and subjected to flashevaporation under a vacuum at 35-40° C. until dry.

A portion of the residue is then chromatographed on a 5×10 cm plate ofsilica gel in solvent B (below) (Merck silica gel 0.25 mm). Authenticstandards of taxol, baccatin, and cephalomannine are alsochromatographed for comparison.

The aqueous layer is lyophilized and thoroughly extracted withchloroform methanol 1:1. When evaporated, it is chromatographed in thesame manner as above.

The standard solvents used in the chromatographic procedures are:

A) chloroform/methanol 7:1 V/V

B) chloroform/acetonitrile 7:3 V/V

C) ethyl acetate/isopropanol 95:5 V/V

D) methylene dichloride/tetrahydrofuran 6:2 V/V.

The taxanes (taxol, baccatin, cephalomannine) all absorb short wave UVlight (254 angstroms) and react with the vanillin-sulfuric acid spray(see, Cardellina, J. Liquid Chromatography 14: p 6659-665 (1991) toproduce an intense blue coloration fading to gray then turning brownafter 24 hours.

Each fungus culture was treated in the same manner. At least 50 fungiand 10 bacteria were isolated from yew roots, needles, stems, or fruitsand tested. Samples of the extract from each organism were subjected toinitial screening by thin layer chromatography (TLC) in solvent A.

A few microbes appeared promising after the initial screening. That is,their extract produced a blue spot at R_(F) 0.75-0.81 in solvent A (sameas taxol). These microbes were fungi and one later identified asCladosporium macrocarpum, was further checked for taxol by othermethods--but without success. A strain of fungus, designated Montana BA,exhibited several important absorbances in HNMR consistent with thespectrum of taxol (see FIG. 1). Therefore, this culture held promise fortaxol production and was studied further. The following are descriptionsof the fungus and taxane production.

(1) Montana BA--rapidly growing on potato dextrose agar, most hyphaewith growth oppressed to agar surface, no apparent fruiting structures,beige coloration of hyphae. (Culture on deposit with the Centraal Bureauvoor Schimmelcultures under the terms of the Budapest Treaty asAccession No. CBS 279.92).

(2) Evidence for taxol (taxane) production

(a) The fungus was grown in both M-1-D and Taxol Microbial culturemedium (2 liters) for 3 weeks at 25° C. with only periodic shaking. Themedium and the grown mycelium were extracted with chloroform:MeOH 10:1v/v.

After evaporation of the chloroform:MeOH the residue was taken up in 0.5ml of CHCl MeOH 10:1 v/v and subjected to preparative TLC in solvent Bon Merck plates 0.5 mm (20×20). A band at R_(F) 0.47-0.50 that hadslight UV (254 angstroms) absorbance and gave a slight reaction with thevanillin sulfuric acid spray was scraped from the plate and eluted withacetonitrile. Only the extreme edge is sprayed, unadulterated materialis scraped and eluted.

Further chromatography was done in solvent A in TLC (precleaned plateswith the same solvent). The "taxol" band was eluted and subjected tospectroscopy. This band had the same FAB mass spectrum as authentictaxol with an M⁺¹ at 854 and prominent peaks at 491, 509 and 613 (seeFIG. 2).

The UV spectrum of the compound had an absorption maximum at 273 nmwhich is identical to authentic taxol (see Wani et al). The NMR spectrumof the semi-purified compound possessed all of the major absorbances ofauthentic taxol (FIG. 1).

In addition, the R_(F) values for the taxol preparation from the fungus"Montana BA" was identical to authentic taxol (see Table 3 below).

                  TABLE 3                                                         ______________________________________                                        Comparative R.sub.F values of fungal taxol to authentic                         taxol and baccatin                                                            Solvent System:                                                             ______________________________________                                                    CHCl.sub.3 /MeOH 78:1 v/v                                           taxol .81                                                                     fungal .81                                                                    baccatin .75                                                                  fungal baccatin .75                                                            CHCl.sub.3 Acetonitrile 7:3 v/v                                              taxol .47                                                                     fungal taxol .47                                                              baccatin .50                                                                  fungal baccatin .50                                                            Ethyl acetate isopropanol 95:5 v/v                                           taxol .63                                                                     fungal taxol .63                                                              baccatin .58                                                                  fungal baccatin .58                                                            Ch.sub.2 Cl.sub.2 Tetrahydrofuran 6:2 v/v                                    taxol .75                                                                     fungal taxol .75                                                              baccatin .67                                                                  fungal baccatin .67                                                         ______________________________________                                    

It is also to be noted that the compound "baccatin", a taxane related totaxol, also appeared in the fungal extract. It, along with taxol,yielded the same intense blue color reaction and the same UV (254)absorption properties as the authentic compounds. It also had the sameR_(F) s as authentic baccatin (Table 3).

                  TABLE 4                                                         ______________________________________                                        Enhanced Taxol Microbial Culture Medium                                                          Grams/Liter                                                ______________________________________                                        glucose            1                                                            fructose 3                                                                    sucrose 6                                                                     KHPO.sub.4 --KH.sub.2 PO.sub.4 1 ml of 1M pH 6.8                              MgSO.sub.4 .36                                                                Ca(NO.sub.3).sub.2 H.sub.2 O .65                                              Yeast extract 0.5 g                                                         ______________________________________                                          Ca(NO.sub.3).sub.2 1.0 mg                                                     ZnSO.sub.4 2.5                                                                MnCl.sub.2 0.5                                                                FeCl.sub.2 2.0                                                                phenylalanine 5.0 mg                                                          NaAc 1.0 g                                                                    Sodium Benzoate 10 mg-100 mg                                                ______________________________________                                    

The amount of taxol per liter (3 wk old culture) is estimated at about1-2 μg. Baccatin appears at a level of 0.5-1 μg/liter.

The taxonomy and properties of the microbe revealed a fungus of thefamily Fungi imperfecti (or alternatively called the family Hyphomyces).The genus was determined to be Taxomyces and the species was namedandreanae.

T. andreanae (Montana BA) forms the bulbil or sterile cell masses whichappear to be unique structures.

Type species: Taxomyces andreanae Strobel, Stierle, & Hess.

Holotypus: Based on material taken from the bark of Taxus brevifoliaNutt. infested living bark samples, agar slants containing the type aredeposited with the Montana State University (MSU) mycologicalcollection, D. E. Mathre, Department of Plant Pathology, Montana StateUniversity--col. no. 738. Duplicate cultures are deposited at the CBS,Baarn, Holland 25 culture 279.92.

A pure culture species was obtained of Montana BA which was namedTaxomyces andreanae S1. Two further fungal cultures, other than MontanaBA, which produced taxanes were obtained from the above screening. Theircharacteristics revealed them to be of the same genus and species, butto have slightly different properties in that each strain differed inhyphal morphology and growth. These microbes were isolated to pureculture form and given the names Taxomyces andreanae 52 and Taxomycesandreanae 53, respectively. Further details are presented below in theexperimental section.

(b) To dispel the notion that the taxol isolated from the culture mediummight be occurring as a result of the fungus's previous association withthe yew tree, the fungus "plugs" used to inoculate the medium wereexhaustively extracted and the residue chromatographed. There was noevidence of taxol.

(c) In order to be assured that taxol is produced "de novo" by thefungus, the fungus was incubated for 3 weeks in two 500 ml cultures ofTaxol Microbial Culture medium and added 100 μCi of NaAC-1-¹⁴ C (54mCi/mole) plus 125 μCi of mixed amino acids 1.75 mCi/mg. Both the taxoland baccatin areas on the preparative TLC's were isolated and thensubjected on 2 plates to 3 dimensional TLC (20×20 plate--25 mm layer)along with (co-chromatography) authentic taxol and baccatin. The UVabsorbing spot was removed by scraping and counted by liquidscintillation counting methods. The results show that the area on theplates having radioactivity was identical to the UV absorbing spot. Thiswas true on both taxol and baccatin. This test assures that bothbaccatin and taxol were synthesized `de novo` by the fungus.

Throughout the course of the taxol microbial study, a means ofdetermining the cytotoxicity of the culture extracts and various columnfractions to facilitate bioassay guided fractionation was needed.Ferrigni et al., J. Nat. Prod. 45, 679 (1982); and Ferrigni et al., J.Nat. Prod. 47, 347 (1984), suggested that a simple brine shrimp assayprovided a reasonable facsimile of the standard anticancer assays. Thoseauthors were able to isolate the antileukemic principles from the seedsof Euphorbia lagascae Spreng using a fractionation scheme guided bybrine shrimp and potato disc assays. Several studies have demonstratedgood correlation between brine shrimp lethality and cytotoxicity.

The brine shrimp bioassay is a simple test. Brine shrimp are hatched inInstant Ocean dissolved in tap water, which generates a solution thatapproximates the constituents of sea water. The test material is handledaccording to its polarity and purity. Crude culture residues are testedat 10 mg, simply dissolved in the saline water used to rear the brineshrimp. The test material is dissolved in 2 ml of salt water, and anadditional 2 ml of salt water containing the brine shrimp is added. Thebrine shrimp are counted, then recounted at various time intervals.Toxicity is determined by the number of brine shrimp deaths which resultfrom the test material, relative to a control of saline water, and acontrol of authentic taxol.

Biological activity of the Montana BA extract showed positive α-action(antitubulin) activity which is the comparable activity observed forauthentic taxol (see references). Other biological activities observedwere a positive brine shrimp test.

The members of the genus/species Taxomyces andreanae were cultivatedwith labeled nutrients to show definitively that the microbes producedtaxanes. It was determined that the addition of benzoic acid to theculture medium increased the amount of taxol and other taxanes.

The present invention is directed to any microbe, especially fungi,which have taxane producing characteristics, especially as described inthe present invention, irrespective of their source. Such microbes arethose which can produce taxol by the NMR spectrum of FIG. 1, the FABmass spectrum of FIG. 2, the SEMS of FIGS. 4, 5 and 6 and the TEM ofFIGS. 8 and 9.

In another aspect of the invention, referred to above, an appropriatelylabelled precursor is used to produce labelled taxol. ¹⁴ C-phenylalanineis the preferred amino acid precursor, for ¹⁴ C-taxol production.However, as in the above example, Na acetate-l-¹⁴ C can be used becauseof its relatively low price and ability to label taxol uniformly.

The taxanes, e.g., taxol, or radiolabelled taxanes produced according tothe present invention, can also be provided as a pharmaceuticalcomposition in combination with one or more pharmaceutically acceptableinert or physiologically active diluents or adjuvants. Thesecompositions may be prepared in any form appropriate for theadministration route desired. The parenteral route and especially theintravenous route are preferred methods of administration. Compositionsfor parenteral administration may be aqueous or nonaqueous sterilesolutions, suspension or emulsions. Propylene glycol, vegetable oils,injectable organic esters and the like, may be used as solvents orvehicles. The compositions may also contain adjuvants, wetting agents,emulsifiers or dispersants.

The compositions may also be in the form of sterile solid solutionswhich may be dissolved or dispersed in sterile water or any injectablesterile medium. The pharmaceutical compositions may be particularly usedin the treatment of acute leukemia and solid tumors at doses known tothe art, but generally in the range of between one and two milligramsper kilogram of body weight by the intravenous route for an adult. Thepharmaceutical compositions should contain about 0.001 to 1.0 wt. % ofeffective ingredient and administered in dosage amounts known to the artfor taxol.

The above microbial culture method allows for bulk compositionscomprising amounts of taxanes, i.e., taxol, in bulk quantities notpreviously available. Previously, only small amounts of taxol, e.g.,only a few hundred milligrams, have been available from extraction andother methods.

An example of a taxol microbial culture medium is set forth below intable 5.

                  TABLE 5                                                         ______________________________________                                        Taxol Microbial Culture Medium                                                  (high sugars)                                                               ______________________________________                                                              8/liter                                                 ______________________________________                                          glucose 1                                                                     fructose 3                                                                    sucrose 6                                                                     Na acetate 1                                                                  casein amino acids 0.5                                                        KH.sub.2 PO.sub.4 pH 6.8 1 ml of 1M                                         ______________________________________                                           mg/liter                                                                   ______________________________________                                          vitamins                                                                      thiamine 1                                                                    biotin 1                                                                      pyridoxal 1                                                                   calcium pantothenate 1                                                        sodium benzoate 10-100                                                        MgSO.sub.4 3.6                                                                Ca(NO.sub.3).sub.2 6.5                                                        Ca(NO.sub.3).sub.2 1                                                          ZnSO.sub.4 2.5                                                                MnCl.sub.2 5                                                                  FeCl.sub.2 2                                                                ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Growth and bulbil formation of Taxomyces andreanae                              on various tree species normally growing in the vicinity                      of Taxus brevifolia. All observations were recorded 1                         week after inoculation with a 1.0 × 1.0 agar block                      supporting fungal growth.                                                              Av growth                                                            from edge of                                                                  agar block Formation of                                                       (cm) bulbils*                                                                          Twigs/             Twigs/                                            Plant Species leaves Bark leaves Bark                                       ______________________________________                                        Taxus brevifolia                                                                         2.0      0.25-0.5  heavy   heavy                                     Nutt.                                                                         Pacific Yew                                                                   Betula nigra L. 1.0 0.75 heavy moderate                                       River Birch                                                                   Pinus monticola 0.1 0.2 light light                                           Dougl.                                                                        Western white, pine                                                           Tsuga heterophylia 0.0 0.1 none none                                          Rafn Sarg.                                                                    Western Hemlock                                                               Pseudotsuga 0.1 0 none none                                                   taxifolia                                                                     (Poir) Britt.                                                                 Douglas fir                                                                   Thuja plicata Donn 0.5 0.5 light to heavy                                     Western Red Cedar   moderate                                                  Picea engelinanni 0.1 0 moderate none                                         Parry ex Engelrn.                                                             Engelmann spruce                                                              Larix occidentalis 0 0 none none                                              Nutt.                                                                         Western larch                                                               ______________________________________                                         *Bulbil formation is given in terms of heavy (completely covering the are     where mycelium is growing) to moderate, to light (little or sparse bulbil     formation).                                                              

The fungi according to the present invention, when grown in a definedmedium, use sodium benzoate 10-100 mgs/liter to recover the taxol fromthe medium. The fungi are preferably maintained as an inoculum source ina freshly prepared malt agar (100 mgs of benzoate/liter). The fungi donot grow well on shake culture. The optimal fungal growth occurs onbactosoytone 5-7 grams/liter (instead of peptone include the essentialamino acids and fructose and glucose). With 10 grams sucrose, togetherwith benzoate, vitamins and minerals as per Taxol Microbial culturemedium. To further confirm the presence of taxol and baccatin in fungalpreparations and extract, a culture of T. Andreanae was prepared byCHCl₃ /MeOH extraction, prepared TLC on CHCl₃ /acetonitrile 7:3 v/vfollowed by elution of a 1 centimeter wide area at R_(F) 0.18-0.25. Thenthe residue was subjected to a micropore HPLC separation from optimumsensitivity. The study was conducted on LC/MS using a reverse phasecolumn (1 mm)×150 mm×5 μm particles with an Isocratic mobile phaseconsisting of 65 percent acetonitrile/35 percent 2 mM ammonium acetateat a flow rate of 50 microliters/minute. Subsequent analysis of twomicroliters of the sample prepared from 100 microliters of the dissolvedsample prepared from 100 microliters of the dissolved sample yielded apeak with the retention time identical to taxol (7.85 minutes) and 6.4minutes consistent with the pseudomolecular ion (M+NH₄)⁺ baccatin III.

FIG. 3 illustrates the mass spectrum of the taxol eluting peak at854-871 consistent with taxol. The amount of taxol produced in thisparticular medium is approximately 1 microgram/liter of the medium.

This does not represent endogenous taxol from the yew tree for severalreasons. Radiolabeling experiments with phenylalanine, sodium benzoateor acetate ¹⁴ C as precursors yields radiolabelled taxol. Accordingly,the experiments clearly show that the taxol is being synthesized.

To further describe the Taxomyces andreanae microbe, the followingdetailed examples are provided.

EXAMPLE 1

Taxomyces andreanae is a novel endophytic fungus associated with theinner bark of Taxus brevifolia Nutt. (Pacific Yew). This fungus hassmall hyphae which average 1.2 μm in diameter. It characteristicallyforms clumps of loosely constructed cells (bulbil-like). These clumps ofvarious shapes and sizes typically range from about 5×5 to 16×30 μm indiameter and length. The cells in these clumps average about 1.5×2.5 μmand appear to be loosely packed in the bulbi and are incapable ofgermination. This fungi grows rapidly on many common laboratory media,covering the plates with its mycelium in three or four days. It lacksclamp connections and dolipore septations. Its telemorph is unknown. TheTaxomyces andreanae is a endophytic hyphomycete, isolated exclusivelyfrom the inner bark on small limbs of a specific yew tree in northernMontana.

The fungus species were isolated from the upper limbs of a shrub-likePacific yew tree growing as undercover in a mature undisturbed cedarforest in Flathead County, Montana. Small limbs (0.5-1.0 centimeter)were surface treated with 70 percent ethanol. The outer bark was peeledback and pieces of the white inner barks (phloem/cambium) wereaseptically removed and placed on H₂ O agar. Hyphal tips of fungigrowing from the pieces of the plant were placed on mycological agar andfungal growth was enhanced. The growth pattern of the fungus was studiedon other plant species. Leaf, stem, and bark samples of various plantspecies growing near the tree from which this fungus was obtained werecollected near the Hungry Horse dam site in the Flathead NationalForest. Small pieces (0.5-2 cm) of leaves, stems and barks of thesespecies were placed over four layers of cheesecloth, thoroughlydampened, and autoclaved. Agar blocks (1.0×1.0 cm) supporting fungalgrowth were then placed on the sterilized plant material and fungalgrowth was observed and measured after one week. The growth of thefungus was measured daily after placement of 0.5×0.5 cm agar blocks onstandard freshly prepared agar media plates (Difco), e.t., potatodextrose broth agar, nutrient agar, oatmeal agar, cornmeal agar, limabean agar, water agar, and malt agar.

EXAMPLE 2

Agar blocks having mycelia and bulbil-like structures were fixed anddehydrated as for transmission and scanning electron microscopy (SEM)(FIGS. 6, 7, 8, and 9). For SEM, the material was then critical pointdried, gold coated and sputter coated, and observed with a JEOL 840Ascanning electron microscope. Fungal structures were measured on SEMmicrographs after critical point drying of tissues.

This drying procedure caused some shrinkage of biological structures(about 10 percent) which means that they were probably slightly larger,and the clumps of cells more tightly packed than in the living state.

EXAMPLE 3

Taxonomic Treatment and Description Taxomyces andreanae S1: Strobel,Stierle and Hess gen. et sp. nov. (FIGS. 4-9).

A pure culture species was obtained of Montana BA which was namedTaxomyces andreanae S1. This microbe producing taxane was obtained fromthe above screening. Its structures are described as follows.

Fungus endophyticus e cortice interiora Taxo brevifolo Nutt.; hyphaedimporphae--parvae 1.25 μm et magnae ca 3.75 μm latae et longae;bulbilus cellularum ca 1.25×2.5 μm et laxe continguus et apparenter nongerminans; mycelium celiter crescens, hyphis fibulis nullis et doliporisseptis nullis; telemorphus ignotus.

Mycelium superficial, composed of a network of highly branched, septate,usually hyaline, smooth walled hyphae. Smaller hyphal cells average 1.25μm in diameter. Larger cells average 3.75 μm in diameter (FIGS. 4-5).Cells are budded from fructigenous hyphae forming clumps which varyenormously in shape from spherical to ovoid to longiform and in sizefrom 5×5 μm in length (for elongate bulbils). Bulbil cells remainingcolorless. The cells seem to be loosely packed in the bulbil and areovoid ca. 1.5×2.5 μm and are never observed to germinate.

The "clumps" of cells in FIGS. 5 and 6 are not located on sterigmata,but seem to arise by a "budding process" (FIG. 6). Number of cells ineach clump varies widely (FIGS. 5 and 6). These "clumps" are referred toas bulbils after the broad definition of deBary (Comparative Morphologyand Biology of the Fungi, Mycetozoa and Bacteris, "English Translation,Clarendon Press, Oxford), that is, "small pluricellular bodies incapableof germination". In T. andreanae, the cells of the bulbils, unlike mostbulbilliferous fungi, appear to be loosely packed, but neverthelessconnected with fibrous material (FIGS. 6, 9). These clumps of cellsmight also be considered as conidial masses but since germination hasnever been observed (in sterile H₂ O and nutrient broth), the clumps ofcells seem to better fit the broad description of a bulbil.

Furthermore, transmission electron microscopic examination of thesebulbil cells reveals that they are engorged with cytoplasmic structuresincluding lipid bodies (FIGS. 8, 9). They also possess a bilayered cellwall (FIGS. 8, 9). Nevertheless, these bulbils differ from the bulbilsof other standard bulbilliferous fungi by lacking pigmentation, certainsclerotial-like qualities (outer rind-like cells and inner swollencells) and in the manner of their formation.

EXAMPLE 4

A pure culture species was obtained which was named Taxomyces andreanaeS2. The fungus species was isolated from the upper limbs of a shrub-likePacific yew tree growing as undercover in a mature undisturbed cedarforest in Flathead County, Montana as described in Example 1, above.Small limbs (0.5-1.0 centimeter) were surface treated with 70 percentethanol. The outer bark was peeled back and pieces of the white innerbarks (phloem/cambium) were aseptically removed and placed on H₂ O agar.Hyphal tips of fungi growing from the pieces of the plant were placed onmycological agar and fungal growth was enhanced.

The growth pattern of the fungus was studied on other additional plantspecies. Leaf, stem, and bark samples of various plant species growingnear the tree from which this fungus was obtained were collected nearthe Hungry Horse dam site on the Flathead National Forest. Small pieces(0.5-2 cm) of leaves, stems and barks of these species were placed overfour layers of cheesecloth, thoroughly dampened, and autoclaved. Agarblocks (1.0×1.0 cm) supporting fungal growth were then placed on thesterilized plant material and fungal growth was observed and measuredafter one week. The growth of the fungus was measured daily afterplacement of 0.5×0.5 cm agar blocks on standard freshly prepared agarmedia plates (Difco, e.g., potato dextrose broth agar, nutrient agar,oatmeal agar, cornmeal agar, lima bean agar, water agar, and malt agar.

The fungal culture of Taxomyces andreanae S2, was tested and found toproduce taxanes by the procedures described above.

EXAMPLE 5

A pure culture species was obtained which was named Taxomyces andreanaeS3. The fungus species was isolated from the upper limbs of a shrub-likePacific yew tree growing as undercover in a mature undisturbed cedarforest in Flathead County, Montana as described in Example 1, above.Small limbs (0.5-1.0 centimeter) were surface treated with 70 percentethanol. The outer bark was peeled back and pieces of the white innerbarks (phloem/cambium) were aseptically removed and placed on H₂ O agar.Hyphal tips of fungi growing from the pieces of the plant were placed onmycological agar and fungal growth was enhanced.

The growth pattern of the fungus was studied on other additional plantspecies. Leaf, stem, and bark samples of various plant species growingnear the tree from which this fungus was obtained were collected nearthe Hungry Horse dam site on the Flathead National Forest. Small pieces(0.5-2 cm) of leaves, stems and barks of these species were placed overfour layers of cheesecloth, thoroughly dampened, and autoclaved. Agarblocks (1.0×1.0 cm) supporting fungal growth were then placed on thesterilized plant material and fungal growth was observed and measuredafter one week. The growth of the fungus was measured daily afterplacement of 0.5×0.5 cm agar blocks on standard freshly prepared agarmedia plates (Difco), e.g., potato dextrose broth agar, nutrient agar,oatmeal agar, cornmeal agar, lima bean agar, water agar, and malt agar.

The fungal culture of Taxomyces andreanae S3, was tested and found toproduce taxanes by the procedures described above.

EXAMPLE 6

Cultural Characterization of Taxomyces andreanae S1

When an agar plug of inoculum of Taxomyces andreanae S1 was placed inthe center of most freshly prepared agar plates enriched with variousnutrients it grew so rapidly that it reached the edge of the plate in 3days (cornmeal agar, lima bean agar, nutrient agar, malt agar, oatmealagar). Bulbils did not form on any of these media up to 6 days afterinoculation. However bulbils were noticed on the inoculum piece on thecornmeal agar after 6 days.

Some bulbils were noticed at the edge of the malt agar plate 7 daysafter inoculation. Numerous fluffy aerial mycelia were especiallyobserved on malt agar and after 6-7 days the mycelium on the malt agardeveloped a deep reddish-brown coloration and a thick mycelial mat.

When the Taxomyces andreanae S1 fungus was placed on the autoclavedleaves, fragments of small limbs and bark taken from various treespecies located in the geographical area of Taxus brevifolia, the bestmycelial growth and bulbil formation occurred on Pacific yew (Table 1,FIG. 4), followed by River birch (Betula nigra) (Table 1). In contrast,there was no growth on, or bulbil formation on Larix occidentalis, orTsuga heterophylla, (Table 1). Other species differentially support weakfungal growth and light bulbil formation, e.g., Pinus monticola, Piceaengelmanni (Table 1) These observations suggest the likelihood that somehost preference of Taxomyces andreanae S1 exists in nature and that itwould be unlikely to be found in and on many species other than Taxus orBetula.

This organism appears to be a saprophyte or endophyte with the latterpreferred since it was found in association with living tissue. There isno evident gross pathology of the host tree. Furthermore, attempts touse agar blocks infested with T. andreanae placed under the bark of yewalso failed to cause any disease manifestation.

Also, the thicker hyphae ca. 3.75 μm in dia, typically extended themycelial mat from one object (leaf or limb fragment or agar block) toanother. These might be considered "exploratory hyphae". Careful studyof the cultural, mycelial and bulbil characteristics in comparison toother bulbilliferous fungi nicely demonstrated the uniqueness ofTaxomyces andreanae.

EXAMPLE 7

A fungal microbe designated as BAC-2BD-1 was isolated from the innerbark of a yew tree and grown on M-1-D medium for three weeks. Obtainedwere dense woolly cream colored mycelium with an irregular spreadingpattern, there were no obvious fruiting. The fungal biotype wasendophytic.

The cultured fungi were ground and extracted with CH₂ Cl₂ /MeOH 10:1.The lipophilic residue was then chromatographed on 0.25 mm silica gelplates (Merck). The chromatography was conducted withchloroform/acetonitrile 7:3 v/v and chloroform/methanol 7:1 v/v. Theresulting plates were examined under UV (254 angstroms) and sprayed with1% vanillin in sulfuric acid. A spot with the same R_(f) and colorreaction (blue fading to brown) identical to taxol appeared.

EXAMPLE 8

A fungi microbe designated as H21 NA was isolated from the needles of ayew tree and grown on M-1-D medium for three weeks. Obtained were fine,translucent, taupe-colored mycelium with an irregular spreading pattern,fruiting structures being present as small brown-rounded bodies. Thefungal biotype was parasitic.

The cultured fungi were ground and extracted with CH₂ Cl₂ /MeOH 10:1.The lipophilic residue was then chromatographed on 0.25 mm silica gelplates (Merck) The chromatography was conducted withchloroform/acetonitrile 7:3 v/v and chloroform/methanol 7:1 v/v. Theresulting plates were examined under UV (254 angstroms) and sprayed with1% vanillin in sulfuric acid. A spot with the same R_(f) and colorreaction (blue fading to brown) identical to taxol appeared.

EXAMPLE 9

A fungal microbe designated as H15 NB was isolated from needles of a yewtree showing spots on the needles. This microbe was grown on M-1-Dmedium for three weeks. Obtained were cream colored fine mycelium, therewere no obvious fruiting structures and the culture was felt-like intexture. The fungal biotype was endophytic or parasitic.

The fungi were ground and extracted with CH₂ Cl₂ /MeOH 10:1. Thelipophilic residue was then chromatographed on 0.25 mm silica gel plates(Merck). The chromatography was conducted with chloroform/acetonitrile7:3 v/v and chloroform/methanol 7:1 v/v.

The resulting plates were examined under UV (254 angstroms) and sprayedwith 1% vanillin in sulfuric acid. A spot with the same R_(f) and colorreaction (blue fading to brown) identical to taxol appeared.

EXAMPLE 10

A fungal microbe designated as 1ND was isolated from the needles of ayew tree, grown on M-1-D medium for three weeks. Obtained were greenmycelium spores identical to cladosporium sp. The fungal biotype wasendophytic.

The fungi were ground and extracted with CH₂ Cl₂ /MeOH 10:1. Thelipophilic residue was then chromatographed on 0.25 mm silica gel plates(Merck). The chromatography was conducted with chloroform/acetonitrile7:3 v/v and chloroform/methanol 7:1 v/v.

The resulting plates were examined under UV (254 angstroms) and sprayedwith 1% vanillin in sulfuric acid. A spot with the same R_(f) and colorreaction (blue fading to brown) identical to taxol appeared.

EXAMPLE 11

A fungal microbe designated as BAC-1NA-1 was isolated from the needlesof a yew tree showing spots on the needles and grown on M-1-D medium forthree weeks. Obtained were velvet-like areas on the older mycelialgrowth and there was no obvious fruiting. The fungi were ground andextracted with CH₂ Cl₂ /MeOH 10:1. The lipophilic residue was thenchromatographed on 0.25 mm silica gel plates (Merck). The chromatographywas conducted with chloroform/acetonitrile 7:3 v/v andchloroform/methanol 7:1 v/v. The resulting plates were examined under UV(254 angstroms) and sprayed with 1% vanillin in sulfuric acid. A spotwith the same R_(f) and color reaction (blue fading to brown) identicalto taxol appeared.

The data set forth in Tables 7-9 indicate additional fungi obtained bythe method according to the present invention. The fungi described bythese data are capable of producing taxol or related taxanes.

Evidence that the fungi obtained by the method of the present inventionproduce taxol includes thin layer chromatographic (TLC) comparisons ofthe organic extracts of these fungi against standard taxol in twosolvent systems, as well as specific monoclonal antibody analysis ofthese extracts.

Of the fungal extracts listed below, extracts 4-73-1, 2, 3, 8, 10, 11,and 12 contained metabolites that behaved in an identical fashion asauthentic (yew) taxol when examined by thin layer chromatography (TLC)on silica gel using the following solvent systems: 95-5 ethylacetate-isopropanol, and 7-3 chloroform-acetonitrile. The organicextracts of 4-73-4, 5, 6, 7, and 9 had compounds that exhibited behaviorsimilar to that of taxol.

The differences in TLC behavior obtained above are also reflected inmonoclonal antibody testing which confirm that the fungal strainsproduce taxol or related taxanes. Hawaii Biotechnology Group, Inc.performed monoclonal antibody testing using Competitive InhibitionEnzyme Immunoassay (CIEIA) with monoclonal antibodies which theydeveloped. The monoclonal antibody used to detect taxol had thedesignation MAB-36C and the monoclonal antibody used for detection oftaxane, in general, had the designation MAB-8A10. Thus, two tests wereperformed which involved an antibody specific to taxol, and an antibodythat reacts with generic taxanes.

The data presented in Tables 7-9 sets forth the actual value of taxoland taxanes detected in a given sample by CIEIA (detected) and the valueextrapolated per liter of culture volume. The data listed for the 4-73series in Table 7 indicates taxol producing fungal strains. The valueslisted under "TOTAL WT." in Table 8 are the same values listed as"Detected" in Table 7.

Also included are the taxol/taxane titers measured for the organicextract of Taxomyces andreanae (TA) (4-58) which was analyzedconcurrently. These values are lower than usual, and probably reflectthe poor growth of this particular batch of the fungus.

All of the data are from fungi grown in media containing bacto-soytone(10 g/L), glucose (30 g/L), sucrose (20 g/L), sodium benzoate (30 mg/L),and sodium acetate (1 g/L). When these fungi were grown in this samemedia to which 2% yew needle broth was added, the taxol/taxane titerswere significantly higher.

The values in Tables 7-9, however, are from extracts which were notadulterated with yew in any way. The production of taxol/taxanessuggested by these data reflects the independent biosyntheticcapabilities of these fungi obtained by the method according to thepresent invention.

                  TABLE 7                                                         ______________________________________                                                 TAXOL        TAXANE                                                                           Extra-         Extra-                                    Detected polated Detected polated                                           CODE FUNGUS (μg/sample) (μg/L) (μg/sample) (μg/L                ______________________________________                                        4-73-1                                                                              CC45BD   0.0031    0.0183 0.4112  2.4306                                  4-73-2 CC50NA1 0.0031 0.0274 0.2099 1.8597                                    4-73-3 CC50NA2 0.0029 0.0246 0.0099 0.0840                                    4-73-4 CC52NC ND ND 0.0069 0.0410                                             4-73-5 CC53NA ND ND 0.0016 0.0350                                             4-73-6 CC53NA1 ND ND 0.0038 0.0274                                            4-73-7 CC53NA2 ND ND 0.0021 0.0144                                            4-73-8 CC53NC 0.0046 0.0254 0.0184 0.1016                                     4-73-9 CC54BA ND ND 0.0043 0.0306                                             4-73-10 CC54BE 0.0032 0.0411 0.0074 0.0950                                    4-73-11 CC57BC2 0.0064 0.1862 0.0078 0.2268                                   4-73-12 CC64BB 0.0035 0.0532 0.0087 0.1352                                    4-58 TA 0.0080 0.0147 0.0593 0.1093                                         ______________________________________                                         Key: ND = not detected                                                        TA = Taxomyces Andreanae                                                 

                  TABLE 8                                                         ______________________________________                                        TAXOL SPECIFIC CIEIA                                                                              ASSAYED                                                      WT. TAXOL  TOTAL WT.                                                         SAMPLE (mg) (μg/ml) N (μg) (WT/WT)%                                   ______________________________________                                        4-62 A       6.8    not detected                                                 B 8.0 0.0093 1 0.0019 0.00002                                                 C 7.9 0.0440 2 0.0088 0.00011                                                 D 7.8 0.0314 1 0.0063 0.00008                                                 E 7.0 0.360 1 0.0072 0.00010                                                  F 1.0 not detected                                                            G 0.6 not detected                                                            H 0.1 not detected                                                           4-61 A 0.8 not detected                                                        B 7.0 0.213 2 0.0043 0.00006                                                  C 7.1 not detected                                                            D 7.6 not detected                                                            E 7.7 not detected                                                            F 8.8 not detected                                                            G 7.7 not detected                                                            H 8.6 not detected                                                           4-58 A 21.0  0.0400 2 0.0080 0.00004                                          4-73 1 4.5 0.0155 1 0.0031 0.00007                                             2 5.0 0.0155 1 0.0031 0.00006                                                 3 4.7 0.0147 1 0.0029 0.00006                                                 4 5.1 not detected                                                            5 5.0 not detected                                                            6 5.3 not detected                                                            7 4.8 not detected                                                            8 4.6 0.0231 1 0.0046 0.00010                                                 9 4.2 not detected                                                            10  5.1 0.0158 1 0.0032 0.00008                                               11A  5.7 0.0320 1 0.0064 0.00011                                              11B  4.8 not detected                                                         12  4.5 0.0173                                                             ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        TAXANE ("generic") CIEIA                                                                          ASSAYED                                                      WT. TAXOL  TOTAL WT.                                                         SAMPLE (mg) (μg/ml) N (μg) (WT/WT)%                                   ______________________________________                                        4-62 A       6.8    not detected                                                 B 8.0 0.0383 1 0.0077 0.00010                                                 C 7.9 0.3595 2 0.0719 0.00091                                                 D 7.8 0.2314 2 0.0463 0.00059                                                 E 7.0 0.0569 2 0.0114 0.00016                                                 F 1.0 not detected                                                            G 0.6 not detected                                                            H 0.1 not detected                                                           4-61 A 0.8 not detected                                                        B 7.0 0.2618 2 0.0564 0.00081                                                 C 7.1 0.0140 1 0.0028 0.00004                                                 D 7.6 not detected                                                            E 7.7 not detected                                                            F 8.8 not detected                                                            G 7.7 not detected                                                            H 8.6 not detected                                                           4-58 A 21.0  0.2965 + 0.075 4 0.0593 0.00028                                  4-73 1 4.5 2.0560 + 0.18  3 0.4112 0.00914                                     2 5.0 1.0496 2 0.2099 0.00420                                                 3 4.7 0.0497 2 0.0099 0.00021                                                 4 5.1 0.0343 + 0.006 3 0.0069 0.00013                                         5 5.0 0.0081 1 0.0016 0.00003                                                 6 5.3 0.0191 2 0.0038 0.00007                                                 7 4.8 0.0105 2 0.0021 0.00004                                                 8 4.6 0.0920 + 0.016 3 0.0184 0.00040                                         9 4.2 0.0215 1 0.0043 0.00010                                                 10  5.1 0.0372 2 0.0074 0.00015                                               11A  5.7 0.0392 2 0.0076 0.00014                                              11B  4.8 0.0205 1 0.0041 0.00009                                              12  4.6 0.0446 2 0.0089 0.00019                                            ______________________________________                                    

A selected collection of taxol producing microorganisms tested above andfurther defined below, including 13 fungi and 1 bacterium were depositedwith the Agricultural Research Service Culture Collection (NRRL), 1815North University Street, Peoria, Ill., 61604, U.S.A. under the terms ofthe Budapest Treaty.

All of the microbes described in this application have been isolatedfrom the bark or needles of the northwest Pacific yew tree, Taxusbrevifolia. Ten of these microbes have been submitted for deposit at theNorthern Regional Research Center (NRRL) in Peoria, Ill. The microbessubmitted represented distinct morphotypes, i.e., only onerepresentative of the Genus Penicillium was submitted for deposit,although others have been detected. These are only examples of microbeswhich produce taxol, others have been obtained by the same methoddescribed herein. The deposited cell lines have also been tested usingCIEIA immunoassay.

All of these microbes have consistently shown evidence of taxol and/ortaxane production based on thin layer chromatography data and repeatedimmunoassay data obtained at both Hawaii Biotechnology Group, Inc. inAieia, Hi., and in an in-house assay at Montana State University.

All of the data accrued has been obtained either from microorganismsgrown in defined synthetic media, containing no yew broth, or in mediato which yew broth has been added. In each data set reported below,media is clearly indicated.

De novo biosynthesis of taxane or taxol by microorganisms 1-14 is shownbelow. The following descriptions include gross morphology, tissue type,biological activity of organic extract of liquid culture of a particularmicrobe grown in mycological broth, and compounds which we have isolatedand identified from these extracts.

Biological activity is determined by the concentration disk method: eachmicrobe is tested against 2 gram(+) and 3 gram(-) bacteria, and 2 fungi.Zones of inhibition listed are indications of activity. Brine shrimpcytotoxicity is ascertained by LD₅₀ of extract towards brine shrimp. "Noactivity" simply indicates that no activity was observed in these testsin the laboratory, not the absolute absence of biological activity.

Fungi: Mold Type

1. 1ND--On M1D (see Table 1) agar: Very fine, highly branched myceliawith tendency to grow into media. Deep bottle-green, velvety appearanceto both conidia and conidiophore. Conidia are ellipsoid, two-celled andcatenulate, and form in unbranched chains, length≈11 μm, with≈6 μm. Doesnot germinate at 37° C. "Illustrated Genera of Imperfect Fungi" Eds. H.L. Barnett and Barry B. Hunter used to key fungus to Order Moniliales,Genus Bispora. isolated from the needles of a shrubby yew tree on Sep.7, 1991. Deposited NRRL Mar. 3, 1994, Accession No. NRRL 21210.Bioactivity: brine shrimp cytotoxic. See FIG. 10.

2. H1RE--On M1D agar: Very fine, highly divided, off-white myceliagrowing into the medium. Dense, thick pale grey conidiophores coverplate within 7 days. Tentatively identified as Genus Penicillium.Isolated from the roots of a healthy shrubby yew tree. Bioactivity:antibacterial, antifungal, brine shrimp cytotoxic. Compounds isolated todate: phomopsolides. Deposited NRRL Mar. 3, 1994, Accession No. NRRL21208.

3. H10BA2--On M1D agar: Very fine, highly branched off-white myceliagrowing down into the medium. Velvety pale green conidiophore withsmooth conidia, 2.4×2.4 μm. Fungus keyed to Genus Penicillium by Dr.Rajinder Siddhu. Isolated from bark of shrubby yew tree. Deposited NRRLMar. 3, 1994, Accession No. NRRL 21209. Bioactivity: antibacterial,antifungal, brine shrimp cytotoxicity. Compounds isolated to date:penitrem A & B. See FIG. 11.

4. CC45BD--On M1D agar: fine, white, branched, septate mycelia, withdark green spores forming after 96 hours. Little or no aerial growth. Onmycological agar: mycelia is denser and tends to aerial growth; greenspores not formed. Isolated from the inner bark of a 26" yew tree.Deposited NRRL Mar. 3, 1994, Accession No. NRRL 21207. Biologicalactivity: antibacterial. See FIG. 13.

5. CC50NA1--On M1D agar: Off-white mycelia with long, intertwining,branched, septate, filamentous hyphae, and irregular edge. LikeTaxomyces andreanae, hyphae appear to be of two different sizes.Consistent aerial growth throughout plate gives a "fuzzy" appearance.White bulbils are formed along the length of the hyphae. Isolated fromhealthy needles of a yew tree. Deposited NRRL Mar. 3, 1994, AccessionNo. NRRL 21204. Biological activity: antibacterial. See FIG. 14.

6. CC50NA2--similar to CC50NA1, but thicker, fuzzier mycelia.

7. CC50NB--On M1D agar: Fine, off-white, highly divided mycelia growinginto the medium. Deep green velvety conidiophore form after 2 days.Smooth conidia: 4.8×4.8 μm; smooth stipe: 24×4.8 μm, with enlargedapice; metula: 9.6-12.0 μm; phialide: 7.2 μm. Keyed to GenusPenicillium. Isolated from healthy needles of a 24" yew tree.Bioactivity: antibacterial, antifungal, brine shrimp cytotoxic.Compounds isolated to date: gliovictin, compactin, griseofulvin,dechlorogriseofulvin, kojic acid.

8. CC52NC--On M1D agar: White, finely divided, highly branched,irregularly edged mycelia with intermittent aerial habit. Aerial hyphaehave short "clusters" at regular intervals. Isolated from the healthyneedles of a 26" yew tree. Deposited NRRL Mar. 3, 1994, Accession No.NRRL 21212. Biological activity: no activity. See FIG. 19.

9. CC53NA2-1--On M1D agar: Cream colored mycelia with dense, velvetypatches of cream to tan, surrounded by deep green, with dark greenspores. Thick aerial pycnidia form in cultures after 2 weeks. These arewhite with a brown core. Tentatively identified as a Xylaria sp. Liquidcultures grown in M1D broth grow quickly and form a thick, syrupy, pinkexudate. Isolated from the needles of a 26" yew tree. Biologicalactivity: no activity. See FIG. 18.

10. CC54BA--On M1D agar: Dense, fuzzy mycelial mat consisting of fine,long, thin, branched septate hyphae with terminal and mid-strandbudding. Turns rich salmon pink after 5 days. Isolated from the innerbark of an 18 inch yew tree. Deposited NRRL Mar. 3, 1994, Accession No.NRRL 21205. Biological activity: slightly antibacterial, brine shrimpcytotoxic. See FIG. 17.

11. CC54BE--On M1D agar: Dense, fuzzy white mycelial mat with long,thin, sepatate, filamentous hyphae with little branching. Isolated fromthe bark of an 18" yew tree. Deposited NRRL Mar. 3, 1994, Accession No.NRRL 21211. Biological activity: slightly antibacterial, brine shrimpcytotoxic. See FIG. 15.

12. CC57BC-1--on M1D agar: Velvety green appearance, typical ofPenicillium sp. The microorganism was isolated from 28" yew tree bark.Bioactivity: antibacterial, antifungal, brine shrimp cytotoxic.Compounds isolated to date: mycophenolic acid.

13. CC64BB--On M1D agar: Fine white mycelial mat consisting of long,thin, branched, septate mycelia. Mycelia develops aerial habit towardsedge of late, forming fluffy white mycelial "clusters" after 5 days ofgrowth. Isolated from 28 " yew tree bark. Deposited NRRL Mar. 3, 1994,Accession No. NRRL 21206. Biological activity: antibacterial, brineshrimp cytotoxic. See FIG. 16.

Bacteria

14. CC48BB--On Tryptic soy agar or penassay antibiotic agar: Creamy,regular colonies, gram (+) cocci. Isolated from 18" yew tree bark.Identified by Dr. Eid Megeed as a Micrococcus sp. Biological activity:antibacterial, brine shrimp cytotoxic.

A. Additional CIEIA testing was performed to conform the production oftaxol or taxane by the microorganisms. See Table 10.

B. The microbes were grown in yew free media to examine the "uninduced"potential of microbes to make taxol/taxanes. Organisms were grown inmycological broth (soytone and glucose only). The culture volume was 1Liter, so volume factor was not necessary for estimation of /Litertiters. However, only a portion of extract was used, and this factor wasincluded in the extrapolation to /Liter titers. See Table 11.

CIEIA DATA

For all of the fungi we determined taxol and taxane titers in term ofug/L and ug/g dry mycelia weight. We only determined titers in ug/L forthe single bacterium, CC48BB.

A. HAWAII BIOTECHNOLOGY GROUP, INC.

Tested microbes grown in mycological broth+2% yew broth, 100 mL each.Taxol and Taxane/L are extrapolated by multiplying/ fraction values by10 if entire extract was tested. If only 1/2 of the extract was tested,then /fraction titer was multiplied by 20 to obtain /L value. The blankmedium, which consisted of 1L of uninoculated mycological broth+2% yewbroth, obviously did not need extrapolation for the /Liter titer.

                                      TABLE 10                                    __________________________________________________________________________                           Specific activity   Specific activity                     taxol/ taxol/ taxol/dry wt. taxane/ taxane/ taxane/dry wt.                   CH.sub.2 Cl.sub.2 ext. fraction (ug) liter (ug/L) mycelia (ug/g)                                                       fraction (ug) liter (ug/L)                                                    mycelia (ug/g)                     __________________________________________________________________________    CC45BD                                                                             0.0027                                                                              0.408 ug                                                                            4.08 ug                                                                             20.40   1.67 ug                                                                             10.67 ug                                                                            53.35                                CC45BB 0.0045g 0.518 5.18  5.63 56.3                                          CC50NA1 0.0045 0.462 4.62 0.56 5.22 52.2 6.29                                 CC50NA2 0.0022 0.880 8.80 2.83 8.96 89.6 28.90                                CC52NC 0.0024 0.758 7.58 25.26 7.96 79.6 265.33                               CC53NA 0.0012 0.622 6.22 1.38 7.27 72.7 16.16                                 CC53NC 0.0020 0.614 6.14 1.06 5.65 56.5 9.74                                  CC54BA 0.0074 (1/2) 0.304 6.08 1.41 4.20 84.0 19.53                           CC54BE 0.0056 (1/2) 0.174 3.48 0.84 1.82 36.4 8.88                            CC57BC2 0.0065 (1/2) 0.192 3.84 1.16 3.59 71.8 21.75                          CC64BB 0.0060 0.264 2.64 1.39 1.87 37.4 19.68                                 blank (H.) 0.0057 0.95 0.95  6.61 6.6                                       __________________________________________________________________________

                                      TABLE 11                                    __________________________________________________________________________                                   Specific activity      Specific activity                                                              CH.sub.2 Cl.sub.2                                                            total taxol/ taxol/                                                           taxol/dry wt.                                                                 taxane/ taxane/dry                                                            wt.                       sample (g) wt (g) sample (ug) Liter (ug/L) mycelia (ug/g) sample (ug)                                                             Liter (ug/L)                                                                  mycelia (ug/g)          __________________________________________________________________________    CC45BD                                                                              0.0045                                                                              0.0266                                                                             0.0031 0.018  0.020    0.4112 2.43   2.80                      CC50NA1 0.0050 0.0443 0.0031 0.027 0.019 0.2099 1.86 1.30                     CC50NA22 0.0047 0.0399 0.0029 0.025 0.266 0.0099 0.840 8.96                   CC52NC 0.0050 0.0303 nd   0.0069 0.042 8.571                                  CC53NA 0.0050 0.1092 nd   0.0016 0.035 0.016                                  CC53NC 0.0040 0.0254 0.0046 0.025 0.029 0.0184 0.102 0.120                    CC54BA 0.0042 0.0299 nd   0.0043 0.031 0.023                                  CC54BE 0.0051 0.0655 0.0032 0.041 0.856 0.0074 0.095 0.081                    CC57BC2 0.0057 0.1658 0.0064 0.186 0.086 0.0078 0.227 0.105                   CC64BB 0.0046 0.0099 0.0035 0.053 0.053 0.0089 0.135 0.135                  __________________________________________________________________________

C. H10BA2 grown in 26L mycological broth without added yew broth.Estimate of 1 L titer includes volume factor. See Table 14.

Thus all of the exemplary taxol or taxane producing microorganisms setforth herein were obtained by the general method of the invention andfound to produce taxane or taxol.

Taxol produced by any of the above microorganisms can be used in thetreatment of cancers, including, but not limited to lung cancer, ovariancancer, breast cancer, prostate cancer and melanoma, as well as others.See for example, Holmes et al., "Phase II trial of taxol, an active drugin the treatment of metastatic breast cancer", J. Natl. Cancer Inst.,Vol. No. 83, No. 24., December 1991, p. 1797-1805; Rowinsky et al.,"Cardiac disturbances during the administration of taxol", J. ClinicalOncol., vol 9, No. 9, September 1991, pp. 1704-1712; Brown et al.,"Phase I trial of taxol given by a 6 hour intravenous infusion", J.Clinical. Oncol., Vol 9, No. 7, p. 1261-67; "Ovarian Cancer", Semin.Surg. Oncol, Vol. 6, No. 6, p. 328-38; Thigpen et al., "Chemotherapy inovarian carcinoma: present role and future prospects, Semin. Oncol., vol16, (4 Suppl. 6), August 1989, p. 58-65; Roytta et al. "Morphologicalstudies on the effect of taxol on cultured human prostatic cancer cells,Prostate 1987, Vol 11 (1), p. 95-106; McGuire et al., "Taxol: a uniqueantineoplastic agent with significant activity in advanced ovarianepitelial neoplasms" Ann Inter. Med. vol 111 (4), Aug. 1989, p. 273-279.and Wiernik et al., "Phase I clinical and pharmacokinetic study oftaxol", Cancer Res., Vol. 47, No. 9, (May 1, 1987) p. 2486-93.

B. MONTANA STATE UNIVERSITY

These results were obtained Fungi were grown in mycological brothwithout any yew added. 1ND was 2L, H10BA2 was 10L, CC50NA1 and CC50NA22were 1L. Appropriate factoras are used to extrapolate to /Liter titers.

                                      TABLE 12                                    __________________________________________________________________________                                   Specific activity      Specific activity                                                              CH.sub.2 Cl.sub.2                                                            total taxol/ taxol/                                                           taxol/dry wt.                                                                 taxane/ taxane/                                                               taxane/dry wt.                                                                 sample (g) wt (g)                                                            sample (ug) Liter                                                             (ug/L) mycelia                                                                (ug/g) sample (ug)                                                            Liter (ug/L)                                                                  mycelia (ug/g)          __________________________________________________________________________    IND   0.0016 g                                                                            0.0463                                                                             0.0029 0.0042 0.001    0.0058 0.0083 0.002                     H10BA2 0.0085 0.9249 0.0015 0.0163 0.002 0.0879 0.9565 0.143                  CC50NA1 0.0142 0.0499 0.0025 0.0088 0.014 0.0067 0.0234 0.036                 CC50NA22 0.0135 0.463 0.00298 0.0102 0.039 0.0074 0.0254 0.102              __________________________________________________________________________

These were tested Fungi were grown in media 1 (soytone, yeast, vitamins)and 2% yew broth in 500 mL cultures.

                                      TABLE 13                                    __________________________________________________________________________                                   Specific activity      Specific activity                                                              CH.sub.2 Cl.sub.2                                                            total taxol/ taxol/                                                           taxol/dry wt.                                                                 taxane/ taxane/                                                               taxane/dry wt.                                                                 sample (g) wt (g)                                                            sample (ug) Liter                                                             (ug/L) mycelia                                                                (ug/g) sample (ug)                                                            Liter (ug/L)                                                                  mycelia (ug/g)          __________________________________________________________________________    CC50NA22                                                                            0.0046 g                                                                            0.0146 g                                                                           0.0592 ug                                                                            0.376 ug                                                                             0.313    0.282 ug                                                                             1.790 ug                                                                             1.49                      CC53NA1 0.0093 0.0193 0.0327 0.135 0.259 0.241 1.003 1.93                     4BA 0.0083 0.0198 0.0576 0.274 1.827 0.377 1.798 11.99                        H10BA2 0.0071 0.0366 0.0135 0.058 0.009 0.276 1.189 0.175                     CC50NA1 0.0071 0.0137 0.173 0.669 0.372 0.279 1.077 0.598                     Blank 0.0045 0.0045 0.149 0.149  0.307 0.307                                __________________________________________________________________________

Tested -H10BA2 grown in 26L mycological broth without added yew broth.Estimate of 1L titer includes volume factor. Second sample, H10BA2 grownin 1L broth, also without added yew broth.

                                      TABLE 14                                    __________________________________________________________________________                              Specific activity   Specific activity                 CH.sub.2 Cl.sub.2 total taxol/ taxol/ taxol/dry wt. taxane/ taxane/                                                       taxanes/dry wt.                   sample (g) wt (g) sample (ug) Liter (ug/L) mycelia (ug/g) sample (ug)                                                     Liter (ug/L) mycelia            __________________________________________________________________________                                                  (ug/g)                          H10BA2                                                                             0.0114                                                                             1.3142                                                                            0.1389                                                                              0.600 0.16    0.107 0.316 1.4010.244                        H 0.0103 0.0442 0.1284 0.549 0.190                                          __________________________________________________________________________

These examples were Fungi were grown in 4×100 mL media 1 and 1% yewbroth.

                                      TABLE 15                                    __________________________________________________________________________                              Specific activity   Specific activity                 CH.sub.2 Cl.sub.2 total taxol/ taxol/ taxol/dry wt. taxane/ taxane/                                                       taxanes/dry wt.                   sample (g) wt (g) sample (ug) Liter (ug/L) mycelia (ug/g) sample (ug)                                                     Liter (ug/L) mycelia            __________________________________________________________________________                                                  (ug/g)                          CC52NC                                                                             0.0064                                                                             0.0530                                                                            0.0923                                                                              1.910 ug                                                                            2.94    0.2648 ug                                                                           5.48 ug                                                                             8.431                             CC45BD 0.0040 0.0434 0.0236 0.714 0.193 0.1655 5.006 1.353                    Blank 0.0085 0.0090 0.243 0.257                                             __________________________________________________________________________

These examples were 500 mL, media 1 and 1% yew broth.

                                      TABLE 16                                    __________________________________________________________________________                              Specific activity   Specific activity                 CH.sub.2 Cl.sub.2 total taxol/ taxol/ taxol/dry wt. taxane/ taxane/                                                       taxanes/dry wt.                   sample (g) wt (g) sample (ug) Liter (ug/L) mycelia (ug/g) sample (ug)                                                     Liter (ug/L) mycelia            __________________________________________________________________________                                                  (ug/g)                          CC50NB                                                                             0.0060                                                                             0.0977                                                                            0.0063                                                                              0.206 0.089   0.0398                                                                              1.30  0.568                             CC64BB 0.0053 0.0252 0.0243 0.231 0.481 0.0744 0.707 1.473                    IND 0.0102 0.0301 0.0175 0.103 0.057 0.0728 0.425 0.233                       4BA 0.0125 0.0194 0.3396 1.054 7.027 1.1420 3.544 23.63                     __________________________________________________________________________

Cultures were grown in 250 mL trypyic soy broth with 1% yew needle brothadded.

                                      TABLE 17                                    __________________________________________________________________________                              Specific activity   Specific activity                 CH.sub.2 Cl.sub.2 total taxol/ taxol/ taxol/dry wt. taxane/ taxane/                                                       taxanes/dry wt.                   sample (g) wt (g) sample (ug) Liter (ug/L) mycelia (ug/g) sample (ug)                                                     Liter (ug/L) mycelia            __________________________________________________________________________                                                  (ug/g)                          CC48BB                                                                             0.0112                                                                             0.0225                                                                            0.0143                                                                              0.115         0.266 2.138                                    0.0106 0.220 0.0109 0.091  0.245 2.054                                       Blank 0.0055 0.0055 0.0139 0.074                                               0.0066 0.0454 0.0064 0.177  0.007 0.193                                       0.0073 0.0465 0.0038 0.098  0.0051 0.130                                     Blank 0.0040 0.0331 0.0025 0.024  0.007 0.069                               __________________________________________________________________________

All publications cited herein are incorporated by reference herein intheir entireties.

Taxol is administered in acceptable formulations as set forth inRemingtons Pharmaceutical Sciences, 18th Ed., incorporated herein byreference. Taxol formulations may comprises from about 0.01 to 99%taxol, and may preferably be in dosages of about 50 mg/m2 to about 300mg/m2, which is the maximum dose-limiting toxicity for peripheralneuropathy. Dosage times known to those of skill in the art may be used.A 6-hour IV infusion every 21 days is preferred.

Due to the pioneering nature of the present invention, one of skill inthe art readily recognizes that the present invention also encompasses aprocess for isolating a fungus which produces a pharmaceutical productderived from plant material which comprises the steps of:

(a) obtaining tissue fragments from plant material which is the originof said pharmaceutical product;

(b) placing said tissue fragments on agar medium until fungal growthoccurs;

(c) placing fungal hyphae from said fungal growth on mycological agaruntil a culture in pure form is obtained;

(d) transferring said fungal hyphae to a fungal lab growth medium, andgrowing a fungal culture;

(e) removing at least a portion of the culture media containing thefungal culture, thoroughly grinding the mycelium, and adding achromatographic solvent to the mixture;

(f) obtaining a chromatograph of said fungal culture in saidchromatographic solvent to form a solution;

(g) checking the solution for presence of the pharmaceutical ofinterest, and

(h) isolating the fungal cultures which produce the pharmaceuticalproduct.

In addition, the present invention enables a process for obtaining apharmaceutical product derived from plant material which comprises thesteps of:

(a) obtaining tissue fragments from plant material which is the originof the pharmaceutical product;

(b) placing said tissue fragments on agar medium until fungal growthoccurs;

(c) placing fungal hyphae from said fungal growth on mycological agaruntil a culture in pure form is obtained;

(d) transferring said fungal hyphae to a fungal lab growth medium, andgrowing a fungal culture;

(e) removing at least a portion of the culture media containing thefungal culture, thoroughly grinding the mycelium, and adding achromatographic solvent to the mixture;

(f) obtaining a chromatograph of said fungal culture in said solventsolution; and

(g) checking the solution for presence of the pharmaceutical ofinterest.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept andtherefore such adaptations are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. Allpublications cited herein are incorporated by reference in theirentireties. It is to be understood that the phraseology or terminologyemployed herein is for the purpose of description only and not oflimitation.

What is claimed is:
 1. A method for producing a taxane, which comprisesthe steps of:a) exposing a microbe which produces taxol or taxane in abiologically pure culture to a nutrient media capable of supportinggrowth of said microbe, wherein said microbe is a taxane-producingmicroorganism isolated from a tree of the genus Taxus; b) providingculturing conditions for said media containing said microbe, which arecapable of producing growth and reproduction of said microbe; and c)isolating or concentrating said taxane from said culture media or saidmicrobe.
 2. A method for producing a taxane according to claim 1,wherein, said media comprises benzoic acid.
 3. The method according toclaim 2, wherein said microbe is selected from the group consisting of amicrobe having all the identifying characteristics of deposited strainsNRRL-21204, NRRL-21205, NRRL-21206, NRRL-21207, NRRL-21208, NRRL-21209,NRRL-21210, NRRL-21211 and NRRL-21212.
 4. A method according to claim 1,wherein said microorganism is an endophytic fungi.